Biology subjects

Behler, J.

Publications and source records attributed to Behler, J..

3 recordsLinked to original sources

bioRxiv2020

Multi-platforms approach for plasma proteomics: complementarity of Olink PEA technology to mass spectrometry-based protein profiling

The plasma proteome is the ultimate target for biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which is however still difficult to comprehensively access. The high structural complexity of the plasma proteome can be addressed by either a system-wide and unbiased tool such as mass spectrometry (LC-MS/MS) or a highly sensitive targeted immunoassay such as the Proximity Extension Assays (PEA). In order to address relevant differences and important shared characteristics, we tested the performance of LC-MS/MS in data-dependent and -independent acquisition modes and PEA Olink to measure circulating plasma proteins in 173 human plasma samples from a Southern German population-based cohort. We demonstrated the measurement of more than 300 proteins with both LC-MS/MS approaches applied, mainly including high abundance plasma proteins. By the use of the PEA technology, we measured 728 plasma proteins, covering a broad dynamic range with high sensitivity down to pg/ml concentrations. In a next step, we quantified 35 overlapping proteins with all three analytical platforms, verifying the reproducibility of data distributions, measurement correlation and gender-based differential expression. Our work highlights the limitations and the advantages of both, targeted and untargeted approaches, and prove their complementary strengths. We demonstrated a significant gain in proteome coverage depth and subsequent biological insight by platforms combination - a promising approach for future biomarker and mechanistic studies.

biochemistry↗

bioRxiv2020

Specificities of and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups

The majority of bacteria and archaea possess an RNA-guided adaptive and inheritable immune system against viruses and other foreign genetic elements that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. In some bacteria, multiple instances of cas gene-free (orphan) repeat-spacer arrays exist, while additional instances of arrays that are linked to cas gene cassettes are present elsewhere in the genome. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR-Cas repeat-spacer arrays are present, but only two cas gene cassettes plus a Tn7-associated eleventh array are observed. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support a strong tendency for array fragmentation in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our data demonstrate the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

microbiology↗

bioRxiv2018

Conserved accessory proteins encoded with archaeal and bacterial Type III CRISPR-Cas gene cassettes that may specifically modulate, complement or extend interference activity

A study was undertaken to identify conserved proteins that are encoded either within, or directly adjacent to, cas gene cassettes of Type III CRISPR-Cas interference modules. These Type III modules are especially versatile functionally and have been shown to target and degrade dsDNA, ssDNA and ssRNA. In addition, the interference gene cassettes are frequently intertwined with other accessory genes, including genes encoding CARF domains, some of which are likely to be cofunctional. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families. Most archaeal and bacterial Type III modules were seen to be flanked by several accessory genes, around half of which did not encode CARF domains and remain of unknown function. Non-CARF accessory genes were found to be more diverse than their CARF counterparts, encoding nuclease, helicase, protease, ATPase, transporter and transmembrane domains and including a considerable fraction that encoded no known domains. The diversity of non-CARF Type III accessory genes found in this study suggests that additional families exist which remain undetected because of the limited number of annotated genomes currently available. The method employed is scalable for potential application on metagenomic data once automated pipelines for annotation of CRISPR-Cas systems have been developed. All accessory genes found in this study are presented online in a readily accessible and searchable format for researchers to audit their model organism of choice: http://accessory.crispr.dk.

bioinformatics↗